The main objective of the proposed research is to characterize the chromatin structure of a specific eukaryotic transcription unit. The extrachromosomal ribosomal RNA genes (rDNA) in the macronucleus of the ciliated protozoan, Tetrahymena, will be used as a model system. Psoralen crosslinking of the DNA in living cells will give information about the the structure of the rDNA chromatin (rChromatin) in vivo, and nuclease digestion will be used to probe its structure in isolated nuclei. These same techniques will be used to judge the integrity of purified rChromatin. Isolation of the rChromatin will allow us to characterize its protein components and to determine whether some proteins are localized in spacer regions, transcribed regions, or the promoter region of the rDNA. The composition and structure of the rChromatin in states of different transcriptional activity will be compared. In combination with in vitro transcription experiments, these structural studies should allow us to identify the roles of histones, nonhistone proteins, and RNA polymerase in the regulation of rRNA synthesis. The psoralen crosslinking reaction will be used to probe the secondary structure of the RNA precursor of Tetrahymena. The objective is to identify interactions that may be involved in endonucleolytic processing or in excision of the intervening sequence. Also using psoralen crosslinking, we have found a cluster of putative protein-binding sites near the origin of replication of Drosphila melanogaster mitochondrial DNA (mtDNA). Replication forms of the mtDNA will be analyzed to help determine the role of such proteins in the replication process. The generality of such proteins will be determined by extending the study to the mtDNA of mouse, Tetrahymena, and other species of Drosphila.